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Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs
Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor βPDE cDNAs, which were integrated as si...
Autores principales: | , , , , , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2005
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1174897/ https://www.ncbi.nlm.nih.gov/pubmed/16002788 http://dx.doi.org/10.1093/nar/gki686 |
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author | Andrieu-Soler, Charlotte Casas, Mariana Faussat, Anne-Marie Gandolphe, Christelle Doat, Marc Tempé, Denis Giovannangeli, Carine Behar-Cohen, Francine Concordet, Jean-Paul |
author_facet | Andrieu-Soler, Charlotte Casas, Mariana Faussat, Anne-Marie Gandolphe, Christelle Doat, Marc Tempé, Denis Giovannangeli, Carine Behar-Cohen, Francine Concordet, Jean-Paul |
author_sort | Andrieu-Soler, Charlotte |
collection | PubMed |
description | Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor βPDE cDNAs, which were integrated as single copy transgenes at the same genomic site in 293T cells. Interestingly, antisense ONs were superior to sense ONs for one target only, showing that target sequence can by itself impart strand-bias in gene editing. The most efficient ONs were short 25 nt ONs with flanking locked nucleic acids (LNAs), a chemistry that had only been tested for targeted nucleotide mutagenesis in yeast, and 25 nt ONs with phosphorothioate linkages. We showed that LNA-modified ONs mediate dose-dependent target modification and analyzed the importance of LNA position and content. Importantly, when using ONs with flanking LNAs, targeted gene modification was stably transmitted during cell division, which allowed reliable cloning of modified cells, a feature essential for further applications in functional genomics and gene therapy. Finally, we showed that ONs with flanking LNAs aimed at correcting the rd1 stop mutation could promote survival of photoreceptors in retinas of rd1 mutant mice, suggesting that they are also active in vivo. |
format | Text |
id | pubmed-1174897 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2005 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-11748972005-07-11 Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs Andrieu-Soler, Charlotte Casas, Mariana Faussat, Anne-Marie Gandolphe, Christelle Doat, Marc Tempé, Denis Giovannangeli, Carine Behar-Cohen, Francine Concordet, Jean-Paul Nucleic Acids Res Article Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor βPDE cDNAs, which were integrated as single copy transgenes at the same genomic site in 293T cells. Interestingly, antisense ONs were superior to sense ONs for one target only, showing that target sequence can by itself impart strand-bias in gene editing. The most efficient ONs were short 25 nt ONs with flanking locked nucleic acids (LNAs), a chemistry that had only been tested for targeted nucleotide mutagenesis in yeast, and 25 nt ONs with phosphorothioate linkages. We showed that LNA-modified ONs mediate dose-dependent target modification and analyzed the importance of LNA position and content. Importantly, when using ONs with flanking LNAs, targeted gene modification was stably transmitted during cell division, which allowed reliable cloning of modified cells, a feature essential for further applications in functional genomics and gene therapy. Finally, we showed that ONs with flanking LNAs aimed at correcting the rd1 stop mutation could promote survival of photoreceptors in retinas of rd1 mutant mice, suggesting that they are also active in vivo. Oxford University Press 2005 2005-07-07 /pmc/articles/PMC1174897/ /pubmed/16002788 http://dx.doi.org/10.1093/nar/gki686 Text en © The Author 2005. Published by Oxford University Press. All rights reserved |
spellingShingle | Article Andrieu-Soler, Charlotte Casas, Mariana Faussat, Anne-Marie Gandolphe, Christelle Doat, Marc Tempé, Denis Giovannangeli, Carine Behar-Cohen, Francine Concordet, Jean-Paul Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs |
title | Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs |
title_full | Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs |
title_fullStr | Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs |
title_full_unstemmed | Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs |
title_short | Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs |
title_sort | stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking lnas |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1174897/ https://www.ncbi.nlm.nih.gov/pubmed/16002788 http://dx.doi.org/10.1093/nar/gki686 |
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